Liquids, mixtures, solutions and reacting mixtures are often characterized using optical techniques such as spectrophotometry. In order to characterize samples of these liquids, the liquid is usually contained in a vessel referred to as a cell or cuvette, two or more of whose sides are of optical quality and permit the passage of those wavelengths needed to characterize the liquid contained therein. When dealing with very small sample volumes of, for example, from 1 to 2 microliters, it is difficult to create cells or cuvettes small enough to be filled and permit the industry standard 1 cm optical path to be used. It is also difficult and/or time consuming to clean these cells or cuvettes for use with another sample.
As shown in FIG. 1, micro-volume UV/Vis spectrophotometers described, for example, in U.S. Pat. No. 6,628,382 B2 issued to Robertson on Sep. 30, 2003, the disclosure of which is hereby incorporated by reference in its entirety (however, where anything in the incorporated reference contradicts anything stated in the present application, the present application prevails), measure the absorbance of microliter amounts of liquid samples via a sample retention technology which enables containing a liquid sample by its surface tension between surfaces 2 and 7. The liquid sample forms a column 9 between a light receiving sample interface 7 typically coupled to a first optical conduit such as an optical fiber 11, and a light transmitting sample interface 2, which is typically coupled to a second optical conduit such as an optical fiber 6. The upper 2 and lower 7 sample interfaces can be moved in relation to one another to create multiple known path lengths that are typically less than or equal to 1 mm, thereby expanding the dynamic range of the spectrophotometer for a particular sample, as described in U.S. Pat. No. 8,223,338 B2 issued to Robertson et al., on Jul. 17, 2012, the disclosure of which is hereby incorporated by reference in its entirety (however, where anything in the incorporated reference contradicts anything stated in the present application, the present application prevails). Light 3 from a light source coming through the fiber 6 contained in and flush with surface 2 (also referenced herein as the upper sample interface, or first pedestal surface) radiates downward through the liquid sample column 9 and is collected by the fiber 11 in the lower surface 7 (also referenced herein as the second pedestal surface) of the lower sample interface 4 and sent on to the analysis spectrometer for absorbance measurements.
The placement of the liquid sample is achieved by the user manually pipetting a sample (typically a microliter or two) directly onto the lower sample interface. The absorbance of the sample is measured by taking the negative log of the ratio of the amount of light (I0) transmitted through the system in the absence of the sample and the amount of light (I) transmitted through the system when the sample is present in the sampling interface. Under normal conditions, the amount of light transmitted through the system when the sample is present in the sampling interface is directly proportional to the path length and the concentration of the sample, in accordance with the Beer-Lambert law.
As the use of micro-volume spectrophotometers expands and new applications arise, the need to accurately measure sample absorbance at shorter path lengths to accommodate samples with higher light absorbance properties is increasing. Presently available micro-volume UV/Vis spectrophotometers (e.g., NanoDrop™, Thermo Electron Scientific Instruments, Madison Wis.) can establish an absolute measurement path length that is accurate to approximately ±20 μm. Samples with higher light absorbance properties, however, can require measuring absorbance at path lengths as short as 30 μm.
Therefore, there is a need for an improved path length calibration system and method.